4985 SW 74th Court, Miami, FL USA Tel: (1) , Fax: (1) ,

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1 4985 SW 74th Court, Miami, FL USA Tel: (1) , Fax: (1) , Preliminary Interpretation of Human Fecal Pollution ID TM Results Detection and quantification of the fecal Human gene biomarker for Human fecal contamination by real-time quantitative Polymerase Chain Reaction (qpcr) DNA analytical technology Submitter: ESA Date Received: December 11, 2014 Date Reported: December 31, 2014 SM # Client # Approximate Contribution of Human Fecal Pollution in Water Comment Sample SM-4L11013 Station #1 Negative Negative for 2 human fecal biomarkers SM-4L11014 Station #2 Negative Negative for 2 human fecal biomarkers SM-4L11015 Station #3 Negative Negative for 2 human fecal biomarkers SM-4L11016 Station #4 Negative Negative for 2 human fecal biomarkers SM-4L11017 Station #5 Negative Negative for 2 human fecal biomarkers SM-4L11018 Station #1 Negative Negative for 2 human fecal biomarkers SM-4L11019 Station #2 Negative Negative for 2 human fecal biomarkers SM-4L11020 Station #3 Negative Negative for 2 human fecal biomarkers SM-4L11021 Station #4 Negative Negative for 2 human fecal biomarkers SM-4L11022 Station #5 Negative Negative for 2 human fecal biomarkers Limitation of Damages Repayment of Service Price It is agreed that in the event of breach of any warranty or breach of contract, or negligence of Source Molecular Corporation, as well as its agents or representatives, the liability of the company shall be limited to the repayment, to the purchaser (submitter), of the individual analysis price paid by him/her to Source Molecular Corp. The company shall not be liable for any damages, either direct or consequential. Source Molecular Corp. provides analytical services on a PRIME CONTRACT BASIS ONLY. Terms are available upon request. The sample(s) cited in this report may be used for research purposes after an archiving period of 3 months from the date of this report. Research includes, but is not limited to internal validation studies and peer-reviewed research publications. Anonymity of the sample(s), including the exact geographic location will be maintained by assigning an arbitrary internal reference. These anonymous samples will only be grouped by state / province of origin for research purposes. The client must contact Source Molecular in writing within 10 days from the date of this report if he/she does not wish for their submitted sample(s) to be used for any type of future research.

2 4985 SW 74th Court, Miami, FL USA Tel: (1) , Fax: (1) , Human Fecal Pollution ID TM Quantification Detection and quantification of the fecal Human gene biomarker for Human fecal contamination by realtime quantitative Polymerase Chain Reaction (qpcr) DNA analytical technology Submitter: ESA Date Received: December 11, 2014 Date Reported: December 31, 2014 SM # Client # Analysis Requested Species General Marker Quantified* Human Specific Marker Quantified* DNA Analytical Results SM-4L11013 Station #1 Human Bacteroidetes ID 1 Dorei 1.29E+04 ND** Absent SM-4L11014 Station #2 Human Bacteroidetes ID 1 Dorei 1.40E+04 ND** Absent SM-4L11015 Station #3 Human Bacteroidetes ID 1 Dorei 1.69E+04 ND** Absent SM-4L11016 Station #4 Human Bacteroidetes ID 1 Dorei 5.70E+03 ND** Absent SM-4L11017 Station #5 Human Bacteroidetes ID 1 Dorei 5.35E+03 ND** Absent SM-4L11018 Station #1 Human Bacteroidetes ID 2 EPA 1.29E+04 ND** Absent SM-4L11019 Station #2 Human Bacteroidetes ID 2 EPA 1.40E+04 ND** Absent SM-4L11020 Station #3 Human Bacteroidetes ID 2 EPA 1.69E+04 ND** Absent SM-4L11021 Station #4 Human Bacteroidetes ID 2 EPA 5.70E+03 ND** Absent SM-4L11022 Station #5 Human Bacteroidetes ID 2 EPA 5.35E+03 ND** Absent *Numbers reported as copy numbers per 100 ml of water **Non-detect

3 Laboratory Comments Submitter: ESA Report Date: December 31, 2014 Negative Results In sample(s) classified as negative, the human-associated Bacteroidetes gene biomarker(s) was either not detected in test replicates, one replicate was detected at a cycle threshold greater than 35 and the other was not, or one replicate was detected at a cycle threshold less than 35 and the other was not after repeated analysis. It is important to note that a negative result does not mean that the sample does not definitely have human fecal contamination. Only repeated sampling (both during wet and dry sampling events) will enable you to draw more definitive conclusions as to the contributor(s) of fecal pollution. In order to strengthen the result, a negative sample should be analyzed further for human fecal contamination with other DNA analytical tests. A list of human fecal ID tests can be found at Human Fecal Reference Samples The client is encouraged to submit samples from the surrounding wastewater facilities and/or septic systems in order to gain a better understanding of the concentration of the human-associated fecal Bacteroidetes genetic marker as well as the concentration of the general fecal Bacteroidetes genetic marker in the geographic region of interest. A more precise interpretation would be available to the client with the submittal of such baseline samples. Result Interpretations Quantitative results are reported along with interpretations. Interpretations are given as "negative", trace, "minor contributor", "important contributor", or "major contributor" based on the concentration and proportion of the genetic markers found in the water samples. Additional Testing A portion of all samples has been frozen and will be archived for 3 months. The client is encouraged to perform additional tests on the sample(s) for other hosts suspected of contributing to the fecal contamination. A list of available tests can be found at DNA Analytical Method Explanation All reagents, chemicals and apparatuses were verified and inspected beforehand to ensure that no false negatives or positives could be generated. In that regard, positive and negative controls were run to attest the integrity of the analysis. All inspections and controls tested negative for possible extraneous contaminates, including PCR inhibitors. Each submitted water sample was filtered through 0.45 micron membrane filters. Each filter was placed in a separate, sterile 2ml disposable tube containing a unique mix of beads and lysis buffer. The sample was homogenized for 1min and the DNA extracted using the Generite DNA-EZ ST1 extraction kit (GeneRite, NJ), as per manufacturer's protocol. Amplifications were run on an Applied Biosystems StepOnePlus real-time thermal cycler (Applied Biosystems, Foster City, CA) in a final reaction volume of 20ul containing sample extract, forward primer, reverse primer, probe and an optimized buffer. The following thermal cycling parameters were used: 50 C for 2 min, 95 C for 10 min and 40 cycles of 95 C for 15 s and 60 C for 1 min. All assays were run in duplicate. Absolute quantification was achieved by extrapolating genome copy numbers from standard curves generated from serial dilutions of Human specific and generic genomic DNA. For quality control purposes, a positive control consisting of appropriate genomic DNA and a negative control consisting of PCR-grade water were run alongside the sample(s) to ensure a properly functioning reaction and reveal any false negatives or false positives.

4 Human Bacteroidetes ID TM Species: B. dorei The Human Bacteroidetes ID TM Species: B. dorei service targets the species Bacteroides dorei. B. dorei is an anaerobe that is frequently shed from the gastrointestinal tract and isolated from human feces worldwide. It is a newly discovered species that is widely distributed in the USA. 1,2 The human-associated marker DNA sequence is located on the 16S rrna gene of B. dorei. 3 The marker is the microbial source tracking (MST) marker of choice for detecting human fecal pollution due to its exceptional sensitivity and specificity. Internal validations have been conducted on hundreds of sewage, septage, human and animal host fecal samples collected from throughout the U.S and archived in the Source Molecular fecal bank. The marker has also been evaluated in both inland and coastal waters. A recent, comprehensive, multilaboratory MST method evaluation study, exploring the performance of current MST methods, concluded the B. dorei qpcr assay to be the top performing human-associated assay amongst those tested. The success and consistency of this marker in numerous studies around the world 1,3,4 makes the Human Bacteroidetes ID TM Species: B. dorei service the primary service for identifying human fecal pollution at Source Molecular. Fecal Bacteroidetes are considered for several reasons an interesting alternative to more traditional indicator organisms such as E. coli and Enterococci. 5 Since they are strict anaerobes, they are indicative of recent fecal contamination when found in water systems. This is a particularly strong reference point when trying to determine recent outbreaks in fecal pollution. They are also more abundant in feces of warmblooded animals than E. coli and Enterococci. The Human Bacteroidetes ID TM service is designed around the principle that fecal Bacteroidetes are found in large quantities in feces of warm-blooded animals. 3,5,6,7,8 Furthermore, certain strains of Bacteroidetes have been found to be associated with humans. 3,6 As such, these bacterial strains can be used as indicators of human fecal contamination. Accuracy of the results is possible because the method amplifies DNA into a large number of small copies of the gene biomarker of interest. This is accomplished with small pieces of DNA called primers that are complementary and specific to the unique B. dorei DNA sequence. Through a heating process called thermal cycling, the double stranded DNA is denatured, hybridized to the complementary primers and amplified to create many copies of the DNA fragment desired. If the primers are successful in finding a site on the DNA fragment that is specific to the B. dorei DNA sequence, then billions of copies of the DNA fragment will be available and detected in real-time. The accumulation of DNA product is plotted as an amplification curve by the qpcr software. The absence of an amplification curve indicates that the B. dorei gene biomarker is not detected in the water sample because it is either not present or present at concentrations below the analytical detection limit. To strengthen the validity of the results, additional tests targeting other high-ranking, human-associated Bacteroidetes species should be performed, such as Human Bacteroidetes ID TM Species: B. stercoris, Human Bacteroidetes ID TM Species: B. fragilis, and Human Bacteroidetes ID TM Species: B. thetaiotaomicron. 1 Boehm, A., Fuhrman, J., Mrse, R., Grant, S. Tiered approach for identification of a human fecal pollution source at a recreational beach: case study at Avalon Bay, Catalina Island, California. Environ Sci Technol : Bakir, M., Sakamoto, M., Kitahara, M., Matsumoto, M., Benno, Y. Bacteroides dorei sp. nov., isolated from human faeces. Int. J. Syst. Evol. Microbiol : Bernhard, A., Field, K. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rrna. Appl. Environ. Microbiol. 2000b 66: Ahmed, w., Masters, N., Toze, S. Consistency in the host specificity and host sensitivity of the Bacteroides HF183 marker for sewage pollution tracking. Lett. Appl. Microbiol : Scott, T., Rose, J., Jenkins, T., Farrah, S., Lukasik, J. Microbial Source Tracking: Current Methodology and Future Directions. Appl. Environ. Microbiol : Bernhard, A., Field, K. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes. Appl. Environ. Microbiol. 2000a 66: Fogarty, L., Voytek, M. A Comparison of Bacteroides-Prevotella 16S rrna Genetic Markers for Fecal Samples from Different Animal Species. Appl. Environ. Microbiol : Dick, L., Bernhard, A., Brodeur, T., Santo Domingo, J., et al. Host Distributions of Uncultivated Fecal Bacteroidales Bacteria Reveal Genetic

5 Human Bacteroidetes ID TM : EPA Developed Assay The Human Bacteroidetes ID TM : EPA Developed Assay service targets a functional gene biomarker in Bacteroidales-like anaerobic bacteria that is present in high concentrations in the human gut. The U.S Environmental Protection Agency (U.S. EPA) was the first to target the biomarker using quantitative Polymerase Chain Reaction (qpcr) technology in order to detect ground and surface waters impacted by human fecal pollution. 1 Since it's development, the assay has been used succesfully around the U.S to identify fecal pollution originating from human sources, such as sewage and septage wastewaters. The U.S. EPA Developed assay has been shown to be highly associated with human fecal pollution. It has successfully been validated in multiple nationwide studies using at least 300 individual reference fecal material from 22 different animal species known to commonly contaminate environmental waters. 1,2 A reported 99.2% specificity to human fecal material makes this one of the leading assays to confirm the presence of fecal contamination that is of human origin. 1 The Bacteroidales-like bacteria is widely distributed. It was detected in 100% of hundreds of sewage and human reference fecal samples collected from more than 20 human populations, making it highly sensitive. Internal validations have also been conducted on hundreds of wastewater, human and animal host fecal samples archived in the Source Molecular fecal bank. Fecal anaerobic bacteria are considered for several reasons an interesting alternative to more traditional fecal indicator organisms such as E. coli and Enterococci. 3 Since they are strict anaerobes, they are indicative of recent fecal contamination when found in water systems. 3 This is a particularly strong reference point when trying to determine recent outbreaks in fecal pollution. They are also more abundant in feces of warm-blooded animals than E. coli and Enterococci. The Human Bacteroidetes ID TM : EPA Developed Assay service is designed around the principle that fecal Bacteroidales-like bacteria are found in large quantities in feces of warm-blooded animals. 4,5 Furthermore, certain strains have been shown to be associated with humans. 4,5 As such, these bacterial strains can be used as indicators of human fecal contamination. An advantage of the Human Bacteroidetes ID TM service is that the entire portion of water sampled is filtered to concentrate bacteria. As such, this method avoids the randomness effect of culturing and selecting bacterial isolates. This is an advantage for highly contaminated water systems with potential multiple sources of fecal contamination. Accuracy of the results is possible because the method amplifies DNA into a large number of copies of the gene biomarker of interest. This is accomplished with small pieces of DNA called primers that are complementary and specific to the gene biomarker. Through a heating process called thermal cycling, the double stranded DNA is denatured, hybridized to the complementary primers and amplified to create many copies of the DNA fragment. If the primers are successful in finding a site on the DNA fragment that is specific to the human-associated biomarker, billions of copies of the DNA fragment will be available and detected in real-time. The accumulation of DNA product is plotted as an amplification curve by qpcr software. The absence of an amplification curve indicates that the gene biomarker is not detectable in the water sample either because it is not present or present at concentrations below the analytical detection limit. To strengthen the validity of the results, additional tests targeting other high-ranking, human-associated Bacteroidetes species should be performed, such as Human Bacteroidetes ID TM Species: B. dorei, Human Bacteroidetes ID TM Species: B. fragilis, and Human Bacteroidetes ID TM Species: B. stercoris 1 Shanks, O., Kelty, C., Sivaganesan, M., Varma, M. and Haugland, R. Quantitative PCR for Genetic Markers of Human Fecal Pollution. Appl. Environ. Microbiol : Layton, B., Cao, Y., Ebentier, D., Hanley, K., Ballesté, E., Brandão, J., et al. Performance of Human Fecal Anaerobe-Associated PCR-Based Assays in a Multi-Laboratory Method Evaluation Study. Water Research In Press. 3 Scott, T., Rose, J., Jenkins, T., Farrah, S. and Lukasik, J. Microbial Source Tracking: Current Methodology and Future Directions. Appl. Environ. Microbiol : Bernhard, A., Field, K. Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes. Appl. Environ. Microbiol. 2000a 66: Bernhard, A., Field, K. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rrna. Appl. Environ. Microbiol. 2000b 66:

6 General Bacteroidetes ID TM The General Bacteroidetes ID TM service is designed around the principle that general fecal Bacteroidetes are commonly found in the gastrointestinal tract and feces of Humans and warm-blooded animals worldwide. 1,2,3,4 Fecal Bacteroidetes are considered for several reasons an interesting alternative to more traditional indicator organisms such as E. coli and Enterococci. Since they are strict anaerobes, they are unlikely to persist for extended periods of time in oxygenated environments. Their presence in water systems is therefore indicative of recent fecal contamination. 1,2 This is a particularly strong reference point when trying to determine recent outbreaks in fecal pollution. They are also more abundant in feces of warm-blooded animals than E. coli and Enterococci. 1,2 In addition, a strong positive correlation has been observed between qpcr-measured Bacteroidetes and swimming-associated gastrointestinal illness rates (GIIR). 5 As such, testing for general Bacteroidetes can supplement routine E. coli and Enterococci membrane filtration culture methods and enhance water quality testing results. The General Bacteroidetes ID TM service adapts the qpcr assay from the U.S. EPA Method B for the detection and quantification of the16s ribosomal RNA (16S rrna ) target gene from all known Bacteroidales in water. 6 Accuracy of the results is possible because the method amplifies DNA into a large number of small copies of the gene biomarker of interest. This is accomplished with small pieces of DNA called primers that are complementary and specific to the general Bacteroidetes 16S rrna DNA sequence. Through a heating process called thermal cycling, the double stranded DNA is denatured, hybridized to the complementary primers and amplified to create many copies of the DNA fragment desired. If the primers are successful in finding a site on the DNA fragment that is specific to the Bacteroidetes DNA sequence, then billions of copies of the DNA fragment will be available and detected in real-time. The accumulation of DNA product is plotted as an amplification curve by the qpcr software. The absence of an amplification curve indicates that the Bacteroidetes gene biomarker is not detected in the water sample because it is either not present or present at concentrations below the analytical detection limit. Once general Bacteroidetes is found to be present in the water sample, additional tests may be used to determine the specific sources of the fecal pollution. Please call (786) or visit for a list of available tests. 1 Scott, T., Rose, J., Jenkins, T., Farrah, S., Lukasik, J. Microbial Source Tracking: Current Methodology and Future Directions. Appl. Environ. Microbiol : Dick, L. K., Field, K. G. Rapid Estimation of Numbers of Fecal Bacteroidetes by Use of a Quantitative PCR Assay for 16S rrna Genes. Appl. Environ. Microbiol : Bernhard, A., Field, K. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rrna. Appl. Environ. Microbiol. 2000b 66: Dick, L., Bernhard, A., Brodeur, T., Santo Domingo, J., et al. Host Distributions of Uncultivated Fecal Bacteroidales Bacteria Reveal Genetic Markers for Fecal Source Identification. Appl. Environ. Microbiol : Siefring, S., Varma, M., Atikovic, E., Wymer, L., Haugland, R. A. Improved real-time PCR assays for the detection of fecal indicator bacteria in surface waters with different instrument and reagent systems. J. of Water And Health United States Environmental Protection Agency. Method B: Bacteroidales in Water by Quantitative Polymerase Chain Reaction (qpcr) Assay. 2010